CN117327823A - LMTIA primer set for dual detection of bupleurum components in north and south and application - Google Patents
LMTIA primer set for dual detection of bupleurum components in north and south and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biomolecule detection, and particularly relates to an LMTIA primer group, a probe, a kit and application for double detection of bupleurum components. The invention discloses an LMTIA primer group and a probe for double detection of components of bupleurum, which comprise a bupleurum universal F primer, a bupleurum universal B primer, a bupleurum universal LB primer, a bupleurum LF probe and a bupleurum LF probe. The LMTIA primer group and the probe can be used for double detection of the components of the bupleurum chinense and the bupleurum chinense, have high detection speed, high sensitivity and strong specificity, can rapidly, efficiently and accurately identify the components of the bupleurum chinense and the bupleurum chinense in a sample, and can provide a technical means for the authenticity identification of the traditional Chinese medicinal materials in the road.
Description
Technical Field
The invention belongs to the technical field of biomolecule detection, and particularly relates to an LMTIA primer set, a probe, a kit and application for double detection of bupleurum components.
Background
Radix bupleuri is originally carried in Shen nong Ben Cao Jing (Shen nong's herbal), and is named as radix bupleuri from the original name of BIHu to Ben Cao Tu Jing (herbal pattern Jing), has cold nature and slightly bitter taste, enters liver and gallbladder meridians, has the effects of dispelling heat, soothing liver, relieving depression and raising yang qi, and has an application history of over 2000 years. The Chinese pharmacopoeia specifies that Bupleurum is the dry root of Bupleurum chinense (Bupleurum chinense) of Umbelliferae) Bupleurum chinense DC or Bupleurum angustifolium Bupleurum scorzonerifolium Willd. According to different characters, it is known as "Bei Hu" and nan Hu Shen "respectively. The quality of the germplasm has important influence on the quality of medicinal materials, the domestic bupleurum wild resources are less, and the bupleurum is mainly cultivated, so that the bupleurum cultivated germplasm is more and disordered, and the bupleurum characteristics in different cultivation areas have larger difference. The bupleurum root can show quality and drug effect differences under the influences of different producing areas, different growing modes and the like, so that the conditions of confusion of varieties and quality of medicinal plants of the bupleurum root are caused to a certain extent, and the phenomena of mixing, adulteration and the like of the bupleurum root in different producing areas are frequent, the real quality of the bupleurum root is difficult to ensure, and the safety and the effectiveness of clinical medication of the bupleurum root are greatly threatened. Therefore, a scientific, accurate and stable method for identifying the bupleurum chinense and the bupleurum chinense is established, the technology 'short board' in the aspects of authenticity detection such as screening, identification and the like of the traditional Chinese medicinal materials in the road is favorably overcome, the high-quality rapid development of the traditional Chinese medicine industry in China is promoted, and the method has very important practical significance.
The identification method of bupleurum is various, wherein, the gene detection method has the characteristics of short time, strong specificity, high sensitivity and the like, and has great research value. CN107058605a discloses a fluorescent PCR detection primer group, a probe composition, a kit, a detection method and application for identifying the bupleurum chinense and bupleurum chinense simultaneously; CN114350765a discloses a primer for detecting the northern bupleurum root seed gene and a real-time fluorescent quantitative PCR method. The above method based on real-time fluorescence quantitative PCR requires 1-2 hours to complete detection. The Ladder-type melting temperature nucleic acid isothermal amplification technology (Ladder-shape Melting Temperature Isothermal Amplifiction, LMTIA) is a novel nucleic acid isothermal amplification technology, can realize stable amplification of nucleic acid within 30 minutes, and has higher sensitivity and specificity. At present, no report on the detection of the components of bupleurum chinense by using an LMTIA technology exists. The LMTIA technology is applied to the identification of the components of the bupleurum chinense and the bupleurum chinense, and an LMTIA detection method for double detection of the components of the bupleurum chinense and the bupleurum chinense is established, so that the method has very important practical significance.
Disclosure of Invention
The invention aims to adopt the latest LMTIA technology (the technical principle is shown in CN202011105405.3 or PCT/CN 2020/133584), designs LMTIA primers and probes, establishes a rapid detection method for double detection of the components of the bupleurum chinense and bupleurum chinense, and provides technical support for identification of the traditional Chinese medicinal materials in the road. The invention provides an LMTIA primer group and a probe for double detection of components of bupleurum chinense and bupleurum chinense, which are used for detecting the components of bupleurum chinense and solving the problem of adulteration and counterfeiting of traditional Chinese medicinal materials in the road.
The invention is realized by the following technical scheme:
the LMTIA primer group and probe for double detection of the components of the bupleurum chinense and the bupleurum chinense have the following sequences:
CH-F：5’-GCACCTGGGGCGGCTTTTGCTGAGCCCGTTTACTCTGT-3’
CH-B：5’-CCCCAGGTGCGCGCTTTTGGGTAGTCCCGCCTGACC-3’
CH-LB：5’-TCGAACTGTGACCCCA-3’
NanCH-LF：5’-AAGGGTCGCAGTTGCTC-3’
BeiCH-LF：5’-AAGGGTCGATTTTGCTC-3’
NanCH-LF Pr：BHQ1-CAAAGGGTCGA-JOE
BeiCH-LF Pr：BHQ1-CAAAGGGTCGC-FAM。
the kit for double detection of the components of the bupleurum chinense and bupleurum chinense comprises the LMTIA primer group and a probe.
The LMTIA primer group, the probe and the kit are applied to detection of the components of the bupleurum chinense and bupleurum chinense.
A method for double detection of components of bupleuri radix in south and north comprises: and taking a sample to be detected, extracting sample DNA, preparing an LMTIA reaction system by using the LMTIA primer group, the probe or the kit, and placing the prepared LMTIA reaction system into a real-time fluorescence PCR instrument for isothermal amplification. If an exponential curve appears in the amplification result graph, the sample contains the bupleurum components; if the amplification curve does not appear in the amplification result, the sample does not contain the bupleurum components.
Preferably, the isothermal amplification temperature is 63 ℃, and the isothermal amplification time is 20 minutes.
Preferably, the molar ratio of CH-F to CH-B to CH-LB to NanCH-LF/BeiCH-LF to NanCH-LF Pr/BeiCH-LF Pr in the LMTIA reaction system is 8:8:2:2:5.
The invention also provides the application of the LMTIA primer group and the probe for detecting the components of the bupleurum chinense and bupleurum chinense, and the specific technical scheme is as follows: the application in dual detection of the components of the bupleurum chinense and bupleurum chinense.
The beneficial effects of the invention are as follows: the LMTIA primer group and the probe for detecting the components of the bupleurum chinense provided by the invention have good specificity and sensitivity, the detection speed is high, and the detection of the components of the bupleurum chinense and the bupleurum chinense in a sample can be realized under the constant temperature condition.
The LMTIA primer group and the probe have better specificity at 63 ℃.
The absolute sensitivity of the LMTIA primer set and the probe can reach 1 pg/mu L.
The LMTIA primer set and the probe can be used for detecting the components of the bupleurum chinense in medicinal materials.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
Fig. 1: an ITS sequence alignment chart of the bupleurum chinense;
fig. 2: amplifying result diagram of the amplification temperature optimization of the bupleurum root LMTIA;
fig. 3: amplification result diagram of the amplification temperature optimization of the bupleurum chinense LMTIA;
fig. 4: amplifying result graph of repeatability and stability measurement of bupleurum root LMTIA;
fig. 5: amplification result diagram of the repeatability and stability measurement of the bupleurum chinense LMTIA;
fig. 6: amplification result diagram of the specific measurement of the bupleurum root LMTIA;
fig. 7: amplification result diagram of northern bupleurum LMTIA specificity measurement;
fig. 8: amplification result diagram of the absolute sensitivity measurement of the bupleurum root LMTIA;
fig. 9: amplification result diagram of northern bupleurum LMTIA absolute sensitivity measurement;
fig. 10: amplification result diagram of relative sensitivity measurement of bupleurum root LMTIA;
fig. 11: amplification result diagram of relative sensitivity measurement of bupleurum chinense LMTIA;
fig. 12: amplification result graphs of dual detection of the components LMTIA of the bupleurum chinense and bupleurum chinense;
fig. 13: and (3) detecting amplification result graphs of authenticity identification of the bupleurum chinense in different production areas in the market by using the LMTIA.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The sources of reagents used in the present invention are detailed in Table 1.
TABLE 1 Experimental reagents and sources
Example 1 LMTIA primer set, probe design and temperature optimization
(1) LMTIA primer set and probe design: the sequence of difference between the bupleurum and the bupleurum is screened by using the BLAST sequence comparison analysis and taking the rRNA gene spacer sequences (Internal Transcribed Spacer, ITS) of the bupleurum and the bupleurum as target sequences, as shown in figure 1, and the sequence with a ladder-shaped melting temperature is analyzed and selected by Oligo7 software, so that primers 3Plus are used for designing LMTIA primers, LMTIA Primer design is carried out, and a large number of Primer groups and probes (synthesized by general biological systems (Anhui) with the following sequences are obtained through screening, and the specific sequences are shown in Table 2.
TABLE 2 LMTIA primer set and probe sequences
(2) Extraction of template DNA: bupleurum DNA was extracted using a plant genomic DNA extraction kit purchased from Tiangen Biochemical technologies (Beijing) limited. The specific operation steps are shown in the instruction book of the kit.
(3) The temperature of the LMTIA reaction system is optimized:
the reaction system of the LMTIA is shown in Table 3.
TABLE 3 LMTIA reaction system (10. Mu.L)
Wherein, CH-F, CH-B, CH-LB in the LMTIA primer is a general primer for the bupleurum chinense and bupleurum chinense, and corresponding LF primer and probe Pr are needed to be added respectively when the bupleurum chinense and the bupleurum chinense are detected.
PCR octant was taken and the reagents prepared in Table 3 were added to 4 single tubes (the system does not include template DNA, 9. Mu.L in each single tube), respectively, and 1. Mu.L of ddH was added to 2 single tubes 2 O is used as a negative control, 1 mu L of DNA of bupleurum chinense or bupleurum chinense is added into 2 other single tubes, a Gentier 96E full-automatic medical PCR analysis system is used for setting the temperature gradient to 59 ℃, 61 ℃, 63 ℃ and 65 ℃, and the fluorescence signal is collected every 30 seconds, and 40 fluorescence signals are collected in total.
As can be seen from the observation of the temperature-optimized amplification curve of bupleurum root (see figure 2 of the drawings in the specification): the non-specific amplification caused by primer dimer does not exist at the temperature of 4 ℃ of 59 ℃, 61 ℃, 63 ℃ and 65 ℃. Comprehensively considering the amplification efficiency and reproducibility of the amplification curve of the bupleurum chinense at different temperatures, selecting the optimal temperature of 63 ℃ which is LMTIA, and carrying out subsequent determination. Similarly, the observation of the temperature-optimized amplification curve of bupleurum chinense (see figure 3 of the drawings of the specification) can be seen as follows: north Bupleurum chinense does not exist nonspecific amplification caused by primer dimer at the temperature of 4 ℃ of 59 ℃, 61 ℃, 63 ℃ and 65 ℃. Comprehensively considering the amplification efficiency and reproducibility of the amplification curve of the bupleurum chinense at different temperatures, selecting the optimal temperature of 63 ℃ which is LMTIA, and carrying out subsequent determination. In addition, the optimal temperature of 63 ℃ of the North and south Bupleurum is selected as the LMTIA, which is beneficial to the double detection of the North and south Bupleurum components.
EXAMPLE 3 reproducibility and stability of LMTIA primers of the invention
PCR octant was taken and the reagents prepared in Table 3 were added to 32 single tubes (the system does not include template DNA, 9. Mu.L in each single tube), respectively, and 1. Mu.L of ddH was added to 10 single tubes 2 O was used as a negative control, 1. Mu.L of NanChaihu DNA (or North Chaihu DNA) was added to the other 22 single tubes, and a total of 40 fluorescent signals were collected every 30 seconds using a Gentier 96E fully automated medical PCR analysis system set at a temperature of 63 ℃.
As shown in FIGS. 4 and 5, the amplification curve exists in the LMTIA reaction only when the DNA of the bupleurum chinense or the bupleurum chinense is added, the amplification curve has good repeatability, high stability, positive LMTIA reaction and negative LMTIA reaction. Therefore, the established LMTIA detection method for detecting the components of the bupleurum chinense and bupleurum chinense has better repeatability and higher stability.
EXAMPLE 4 specificity of the LMTIA primer of the present invention
PCR octant was taken and the reagents prepared in Table 3 were added to 26 single tubes (the system does not include template DNA, 9. Mu.L in each single tube), respectively, and 1. Mu.L of ddH was added to 2 single tubes 2 O is used as a negative control, and in the other 24 single tubes, respectively adding 1 mu L of bupleurum DNA, qi Baizhi DNA, angelica dahurica DNA, white aconite DNA, red sage root DNA, angelica sinensis DNA, mung bean DNA, soybean DNA, cassava DNA, corn DNA and olive oil DNA, setting two parallel reactions, setting the temperature to 63 ℃ by using a Gentier 96E full-automatic medical PCR analysis system, collecting fluorescent signals every 30 seconds, and collecting 40 fluorescent signals.
As shown in FIGS. 6 and 7, there is a better amplification curve of LMTIA reaction only when DNA of either North or south radix bupleuri is added, and the LMTIA reaction is positive; the other source DNA showed no amplification curve, and the reaction was negative. Therefore, the established LMTIA detection method for detecting the components of the bupleurum chinense and bupleurum chinense has higher specificity.
EXAMPLE 5 sensitivity of LMTIA primers of the invention
(1) Absolute sensitivity: the extracted DNA of Bupleurum chinense and Bupleurum chinense are diluted in gradient to 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L and 100 fg/. Mu.L. PCR octant was taken and the reagents prepared in Table 3 were added to 14 single tubes (the system does not include template DNA, 9. Mu.L in each single tube), respectively, and 1. Mu.L of ddH was added to 2 single tubes 2 O was used as a negative control, 1. Mu.L of 10 ng/. Mu.L of NanBupleurum DNA (or North Bupleurum DNA) was added to 2 individual tubes, 1. Mu.L of 1 ng/. Mu.L of NanBupleurum DNA (or North Bupleurum DNA) was added to 2 individual tubes, 1. Mu.L of 100 pg/. Mu.L of NanBupleurum DNA (or North Bupleurum DNA) was added to 2 individual tubes, 1. Mu.L of 10 pg/. Mu.L of NanBupleurum DNA (or North Bupleurum DNA) was added to 2 individual tubes, and 1. Mu.L of 1 individual tubes was added to 2 individual tubesMu.l of bupleurum DNA (or bupleurum DNA) in pg/mu.l, and 2 single tubes of bupleurum DNA (or bupleurum DNA) in 1 mu.l of 100 fg/mu.l are added respectively. The temperature was set to 63 ℃ using a Gentier 96E full-automatic medical PCR analysis system, with a total of 40 fluorescent signals acquired every 30 seconds.
As shown in FIGS. 8 and 9, the LMTIA amplification curves were more evident at DNA concentrations of 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L and 1 pg/. Mu.L for either the North or south Bupleurum, but no amplification curve was present at DNA concentrations of 100 fg/. Mu.L, since the concentration of 100 fg/. Mu.L exceeded the detection limit of the method, making amplification unstable. Therefore, the absolute sensitivity of the method can reach 1 pg/mu L, and the DNA of the bupleurum chinense or bupleurum chinense extracted from the sample can be detected when reaching 1pg under a reaction system of 10 mu L.
(2) Relative sensitivity: mixing the bupleurum chinense and bupleurum chinense, wherein the mass fractions of the bupleurum chinense are respectively 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% (corresponding to the mass fractions of the bupleurum chinense of 99%, 95%, 90%, 80%, 50%, 20%, 100%, 5% and 1%) and extracting the DNA of the mixed sample (a plant genome DNA extraction kit, a root of the Chinese day, extracted according to the steps in the specification) as template DNA for standby. PCR octant was taken and the reagents prepared in Table 3 were added to 20 single tubes (the system does not include template DNA, 9. Mu.L in each single tube), respectively, and 1. Mu.L of ddH was added to 2 single tubes 2 O is used as a negative control, 1 mu L of DNA with the mass fraction of 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% of that of the bupleurum chinense is added to 18 other single tubes respectively, two parallel reactions are arranged, the temperature is set to 63 ℃ by using a Gentier 96E full-automatic medical PCR analysis system, and fluorescent signals are acquired every 30 seconds and 40 fluorescent signals are acquired in total.
As a result, as shown in FIG. 10, the LMTIA showed a more pronounced amplification curve when 50%, 80%, 90%, 95% and 99% of Nanhui were included in the mixed sample; the LMTIA has amplification curves but is not obvious when the mixed sample contains 5%, 10% and 20% of bupleurum; the LMTIA had no amplification curve when the mixed sample contained 1% of Nanchai. The above results indicate that 1% exceeded the limit of detection of bupleurum chinense by this method. Therefore, the relative sensitivity of the method for detecting the bupleurum root can reach at least 5 percent. Similarly, as shown in FIG. 11, the LMTIA showed more significant amplification curves when the mixed samples contained 20%, 50%, 80%, 90%, 95% and 99% North Bupleurum; the amplification curve of LMTIA was not evident when the mixed sample contained 10% North Bupleurum; the LMTIA had no amplification curve when the mixed samples contained 1% and 5% North Bupleurum. The above results indicate that 5% exceeded the limit of detection of bupleurum chinense by this method. Therefore, the relative sensitivity of the method for detecting the bupleurum chinense can reach at least 10 percent.
EXAMPLE 6 Using the LMTIA primer set and the probe of the present invention for double detection of the ingredients of North and south Bupleurum
PCR octant was taken and the reagents prepared in Table 3 were added to 8 single tubes (the system does not include template DNA, 9. Mu.L in each single tube), respectively, and 1. Mu.L of ddH was added to 2 single tubes 2 O is used as a negative control, 1 mu L of bupleurum root DNA is added into 2 single tubes to be used as a bupleurum root positive control, and 1 mu L of bupleurum root and bupleurum root mixed DNA is added into 2 single tubes. The temperature was set to 63 ℃ using a Gentier 96E full-automatic medical PCR analysis system, with a total of 40 fluorescent signals acquired every 30 seconds.
As shown in FIG. 12, the LMTIA primer set and the probe of the invention have better amplification curves when detecting the mixture of the bupleurum and the bupleurum, and the contained bupleurum and bupleurum components have LMTIA positive reaction respectively and have higher specificity. Therefore, the LMTIA primer set and the probe can be used for double detection of the components of the bupleurum chinense and the bupleurum chinense, namely, the components of the bupleurum chinense and the bupleurum chinense are identified simultaneously.
EXAMPLE 7 use of the LMTIA primer set and the probe of the present invention for authentication of commercial Bupleurum falcatum in different places of production
PCR octant was taken and the reagents prepared in Table 3 were added to 20 single tubes (the system does not include template DNA, 9. Mu.L in each single tube), respectively, and 1. Mu.L of ddH was added to 2 single tubes 2 O was used as a negative control, and 1. Mu.L of bupleurum DNA was added to 2 additional single tubes as bupleurumPositive control, 1 μl of north bupleurum DNA was added to 2 additional single tubes as north bupleurum positive control, 1 μl of commercial south bupleurum DNA from Jiangxi province was added to 2 additional single tubes, 1 μl of commercial south bupleurum DNA from Yunnan province was added to 2 additional single tubes, 1 μl of commercial north bupleurum DNA from Guangxi province was added to 2 additional single tubes, 1 μl of commercial north bupleurum DNA from Henan province was added to 2 additional single tubes, 1 μl of commercial north bupleurum DNA from Shanxi province was added to 2 additional single tubes, 1 μl of commercial north bupleurum DNA from Gansu province was added to 2 additional single tubes, and 1 μl of commercial north bupleurum DNA from Shanxi province was added to 2 additional single tubes. The temperature was set to 63 ℃ using a Gentier 96E full-automatic medical PCR analysis system, with a total of 40 fluorescent signals acquired every 30 seconds.
As shown in FIG. 13, the LMTIA primer set and the probe of the invention simultaneously detect the positive control of the bupleurum chinense and have better amplification curves, and the commercial bupleurum chinense products are found to be genuine products when other producing areas of bupleurum chinense are detected, and the bupleurum chinense components are not contained; commercial B-site and C-site bupleurum root products are pseudo products, and do not contain the components of the North-south bupleurum root; commercial North Bupleurum falcatum, D, E and F are genuine products, and contain no North Bupleurum falcatum components. Therefore, the LMTIA primer group and the probe can be simultaneously used for identifying the authenticity of the bupleurum chinense in different places of sale.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. LMTIA primer group and probe for double detection of bupleurum components, and is characterized in that the primer group has the following sequence:
CH-F：5’-GCACCTGGGGCGGCTTTTGCTGAGCCCGTTTACTCTGT-3’
CH-B：5’-CCCCAGGTGCGCGCTTTTGGGTAGTCCCGCCTGACC-3’
CH-LB：5’-TCGAACTGTGACCCCA-3’
NanCH-LF：5’-AAGGGTCGCAGTTGCTC-3’
BeiCH-LF：5’-AAGGGTCGATTTTGCTC-3’
NanCH-LF Pr：BHQ1-CAAAGGGTCGA-JOE
BeiCH-LF Pr：BHQ1-CAAAGGGTCGC-FAM。
2. the probe of claim 1 is a proofreading enzyme-mediated probe, having a fluorescent group and a quenching group attached to each end.
3. A kit for dual detection of north and south bupleurum components, which is characterized in that: comprising the LMTIA primer set and the probe according to claim 1 and 2.
4. The use of the LMTIA primer set and the probe in detecting the components of the bupleurum chinense and bupleurum chinense according to claim 1 and 2.
5. Use of the kit of claim 3 in detection of components of bupleurum chinense and bupleurum chinense.
6. A method for double detection of components of bupleurum chinense and bupleurum chinense is characterized in that: taking a sample to be detected, extracting sample DNA, preparing an LMTIA reaction system by using the LMTIA primer set, the probe or the kit of claim 1 and 2, and placing the prepared LMTIA reaction system into a real-time fluorescence PCR instrument for isothermal amplification. If an exponential curve appears in the amplification result graph, the sample contains the bupleurum components; if the amplification curve does not appear in the amplification result, the sample does not contain the bupleurum components.
7. The method for double detection of bupleurum components according to claim 6, wherein: the isothermal amplification temperature is 63 ℃, and the isothermal amplification time is 20 minutes.
8. The method for double detection of bupleurum components according to claim 6, wherein: the molar ratio of CH-F to CH-B to CH-LB to NanCH-LF/BeiCH-LF to NanCH-LF Pr/BeiCH-LF Pr in the LMTIA reaction system is 8 to 8 and 2 to 5.
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